Reactivation of codogenic endogenous retroviral (ERV) envelope genes in human endometrial carcinoma and prestages: Emergence of new molecular targets

Abstract

Endometrial carcinoma (EnCa) is the most common invasive gynaecologic carcinoma. Over 85% of EnCa are classified as endometrioid, expressing steroid hormone receptors and mostly involving pathological prestages. Human endogenous retroviruses (ERV) are chromosomally integrated genes, account for about 8% of the human genome and are implicated in the etiology of carcinomas. The majority of ERV envelope (env) coding genes are either not present or not consistently represented between common gene expression microarrays. The aim of this study was to analyse the absolute gene expression of all known 21 ERV env genes including 19 codogenic and two env genes with premature stop codons in EnCa, endometrium as well as in hyperplasia and polyps. For EnCa seven env genes had high expression with >200 mol/ng cDNA (e.g. envH1-3, Syncytin-1, envT), two middle >50 mol/ng cDNA (envFc2, erv-3) and 12 low less than 50 mol/ng cDNA (e.g. Syncytin-2, envV2). Regarding tumor parameters, Syncytin-1 and Syncytin-2 were significantly over-expressed in advanced stage pT2 compared to pT1b. In less differentiated EnCa Syncytin-1, erv-3, envT and envFc2 were significantly over-expressed. Syncytin-1, Syncytin-2 and erv-3 were specific to glandular epithelial cells of polyps, hyperplasia and EnCa using immunohistochemistry. An analysis of 10 patient-matched EnCa with endometrium revealed that the ERV-W 5' long terminal repeat regulating Syncytin-1 was hypomethylated, including the ERE and CRE overlapping MeCP2 sites. Functional analyses showed that 10 env genes were regulated by methylation in EnCa using the RL95-2 cell line. In conclusion, over-expressed env genes could serve as indicators for pathological pre-stages and EnCa.

Figure 1. QPCR of 21 ERV env genes in normal and pathological endometrial tissues

A: Gene expression analysis by qPCR of control endometrium (n=31), endometrial polyp (n=20), endometrial hyperplasia (n=9) and EnCa (n=38) for 21 ERV env genes. *= P:<0.05 and **= P:<0.005. B: Venn blot of all significant env genes is shown from a comparison of all tissues compared to control endometrium. The intersection represents 7 env genes significantly increased in expression in EnCa and prestages (in bold). Syn= Syncytin.

Figure 2. Correlation of ERV env genes with EnCa staging and histology

A: Significant gene expression differences of Syncytin-1, Syncytin-2 (Syn) and envRb according to different EnCa staging (pT). B: Significant gene expression differences of env genes according to differentiation of EnCa (G1-G3). Statistically significance values are shown for both pT staging and G1-3 tumor grading as *= P:<0.05.

Figure 3. Protein localization of Syncytin-1 in normal and pathological endometrial tissues

Immunohistochemistry (IHC) using Syncytin-1 antibody and paraffin embedded tissues from a control endometrium (A-D); a polyp (E-H), and a complex hyperplasia with atypia (I-L) and an EnCa (M-P). Top row of images (A,E, I and M) represent low magnification (bar=100μm). Bottom row of images represent a high magnification (bar=25μm) of the boxed regions from the low magnification representations. Arrows point to specific morphological structures positive for Syncytin-1 protein expression (brown staining): B: surface epithelium; F, G: epithelial gland cells from a polyp, J, K: epithelial glandular cells from a complex hyperplasia tissue with atypia and showing Syncytin-1 cytosolic protein expression as “punctate” foci (“Golgi-like”) localizing para-nuclear or apically at the membrane; N: EnCa stroma (blue) negative for Syncytin-1 expression and O: Syncytin-1 positive expression in tumor epithelial cells (note positive membrane staining). Negative (neg.) controls (D, H, L, P) represent IHC without Syncytin-1 antibody, but with the secondary antibody.

Figure 4. Protein localization of Syncytin-2 and erv-3 in normal and pathological endometrial tissues

Immunohistochemistry (IHC) using Syncytin-2 and erv-3 antibodies and paraffin embedded tissues from a control endometrium (A,B,G,H); a complex hyperplasia with atypia (C,D,I,J) and EnCa (E,F,K,L). Syncytin-2 and erv-3 left column (low magnification) and right column (high magnification) of microscope pictures are shown (bar=50μm). Arrows point to specific morphological structures positive for Syncytin-2 and erv-3 protein expression (brown staining): B: surface epithelium showing a weak focal Syncytin-2 staining of a ciliated tubal epithelial (mucous producing cell); D: Syncytin-2 positive expression in epithelial gland from a complex hyperplasia tissue with atypia; F: Syncytin-2 expression is negative in EnCa stroma (blue) but positive in epithelial glandular tumor cells (note both cytolosic and membrane staining). For erv-3 protein expression arrows show: H: positive expression in epithelial glandular cells of normal epithelium, J: erv-3 positive expression in epithelial gland and stroma cells from a complex hyperplasia tissue with atypia; L: erv-3 expression is negative in EnCa stroma (blue) but positive in epithelial glandular tumor cells.

Figure 5. DNA methylation analysis of ERV-W 5'LTR

A: Top. Schematic ERV-W provirus with 5'LTR-gag-pol-env (=Syncytin-1) -3'LTR. Numbers represent distances in kb. D= non-codogenic genes gag and pol. Middle graph shows the entire 5'LTR with all 20 CpG (black diamonds) and directly below are arrows which correlate with specific CpG within MeCP2 sites along with the overlapping ERE and CRE DNA elements. Also represented at the bottom graph are the components of the 5'LTR (U3-R-U5). Note that the U3 represents the promoter region containing CpG1-5. B: Analysis of each of the 20 CpG for EnCa and patient matched control endometrium (1-10). Numbers at the right represent the overall methylation level in per cent. Open circle = CpG less than 60% methylated, closed circle = >60% methylation. C: Schematic of control and EnCa organized according to decreasing methylation levels of patient tissues 1-10. D. Graph represents qPCR gene expression values in mol/ng cDNA of Syncytin-1 for each patient modified control endometrium and EnCa patient tissues from B & C. Fold induction of Syncytin-1 were for patient #1: 17-fold, #2: 27.6-fold, #3: 38-fold, #4: 12.2-fold. #5: 8.2-fold, #6: 37-fold, #7: 5-fold, #8: 3.1-fold, #9: 25.5-fold, #10: 4.4-fold.

Figure 6. Functional cell culture studies using demethylation agents

A. Gene expression differences of env genes in fold change after treatment with AzadC (AZA) (n=6) and RG108 (n=6). B: Gene expression differences of 9 methyl-DNA binding proteins after treatment with AzadC (AZA) (n=6) and RG108 (n=6) (untreated = UT). * P:<0.05 and **= P:<0.005. C: Changes of luciferase activity using transfection of different cloned plasmids of ERV-W 5'LTR and estradiol (E2) and ERα in RL95-2-ERα(-) (n=6). Insert shows immunoblot for ERα and GAPDH of RL95-2-ERα(-) cells with and without transfection of CMV-ERα. D: Changes of luciferase activity of different ERV-W 5'LTR after AzadC (AZA) (n=6) and FK treatment (n=6) of RL95-2-ERα(-). Meth= prior methylated DNA. Symbols above each column represents the statistical significance to each experiment; e.g. blue symbol = significant to control. * P:<0.05 and **= P:<0.005.